Alpha helix content of G protein alpha subunit is decreased upon activation by receptor mimetics.
To elucidate the mechanism whereby liganded receptor molecules enhance nucleotide exchange of GTP-binding regulatory proteins (G proteins), changes in the secondary structure of the recombinant Gi1 alpha subunit (Gi1alpha) upon binding with receptor mimetics, compound 48/80 and mastoparan, were analyzed by circular dichroism spectroscopy. Compound 48/80 enhanced the initial rate of GTPgammaS binding to soluble Gi1alpha 2.6-fold with an EC50 of 30 microg/ml. With the same EC50, the mimetic decreased the magnitude of ellipticity, which is ascribed to a reduction in alpha helix content of the Gi1alpha by 7%. Likewise, mastoparan also enhanced the rate of GTPgammaS binding by 3.0-fold and decreased the magnitude of ellipticity of Gi1alpha similar to compound 48/80. In corresponding experiments using a K349P-Gi1alpha, a Gi1alpha counterpart of the unc mutant in Gsalpha in which Pro was substituted for Lys349, enhancement of the GTPgammaS binding rate by both activators was quite small. In addition, compound 48/80 showed a negligible effect on the circular dichroism spectrum of the mutant. On the other hand, a proteolytic fragment of Gi1alpha lacking the N-terminal 29 residues was activated and showed decreased ellipticity upon interaction with the compound, as did the wild-type Gi1alpha. Taken together, our results strongly suggest that the activator-induced unwinding of the alpha helix of the G protein alpha subunit is mechanically coupled to the enhanced release of bound GDP from the alpha subunit.
Tanaka
T
T
Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194, Japan.
Kohno
T
T
Kinoshita
S
S
Mukai
H
H
Itoh
H
H
Ohya
M
M
Miyazawa
T
T
Higashijima
T
T
Wakamatsu
K
K
D002942:Circular Dichroism; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D018716:Molecular Mimicry; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism
D011956:Receptors, Cell Surface; D006153:Guanosine Diphosphate; D019204:GTP-Binding Proteins